Involvement of constitutive nitric oxide synthase in ghrelin-induced cytosolic phospholipase A(2) activation in gastric mucosal cell protection against ethanol cytotoxicity.

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is an important regulator of nitric oxide synthase (NOS) and cyclooxygenase (COX) enzyme systems, the products of which are of major significance to the processes of gastric mucosal defense and repair. Here, using primary culture of rat gastric mucosal cells, we report on the mechanism of ghrelin protection against ethanol cytotoxicity. We show that the protective effect of ghrelin was associated with the increase in NO and PGE2 production, and characterized by a marked up-regulation in cytosolic phospholipase A(2) (cPLA(2)) activity and arachidonic acid (AA) release. The loss in countering effect of ghrelin on the ethanol cytotoxicity was attained with constitutive NOS (cNOS) inhibitor, L-NAME, as well as indomethacin and a specific COX-1 inhibitor, SC-560, while specific COX-2 inhibitor, NS-398, and a selective inducible NOS (iNOS) inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced mucosal cell capacity for NO production, cPLA(2) activation, and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2 generation. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA(2) enzyme protein phosphorylation and S-nitrosylation. Preincubation with L-NAME resulted in the inhibition of the ghrelin-induced S-nitrosylation, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA(2) protein phosphorylation as well as S-nitrosylation. The findings demonstrate that ghrelin protection of gastric mucosa against ethanol cytotoxicity involves cNOS-derived NO induction of cPLA(2) activation for the increase in PGE2 synthesis. This activation process apparently includes the cPLA(2) phosphorylation followed by S-nitrosylation.